This is a revised career development proposal, aimed at understanding molecular regulation of the urea cycle. The urea cycle functions in the liver to convert neuro-toxic ammonia into non-toxic urea. One potential strategy to alleviate the effects of high blood ammonia is to increase patients'innate ability to produce urea by raising the expression of urea cycle enzymes. Such interventions require an understanding of the regulation of ureagenesis at the molecular level. The activities of all urea cycle enzymes coordinately change in response to varying nitrogen loads. However, it appears that different mechanisms regulate gene expression of individual urea cycle genes. The goal of this project is to understand the regulation of ureagenesis at the molecular level. The specific aims of this project are: 1) To determine the functional and structural changes in liver NAGS in response to changing dietary protein intake. My hypothesis is that the ratio of two NAGS variants in the liver mitochondria is determined by the organism's nitrogen load. Furthermore, changes of the ratio of two NAGS variants reflect a post- translational mechanism for regulation of hepatic ureagenesis in response to varying nitrogen load. This hypothesis will be tested by measuring the relative amounts of the two NAGS variants in the livers from mice exposed to different nitrogen load, and identification of mitochondrial protease(s) capable of processing NAGS. 2) To identify signaling pathways that respond to changes in nitrogen loads using transcriptional profiling. My hypothesis is that change in nitrogen loads lead to changes in expression levels of signaling molecules that result in either activation or repression of urea cycle genes. This hypothesis will be tested by measuring changes of mRNA levels in response to varying nitrogen load by transcriptional profiling of RNA using Affymetrix microarrays. 3) To identify proteins whose amounts and post- translational modifications change in response to changing nitrogen loads. My hypothesis is that changes in nitrogen loads triggers changes in amounts and/or post-translational modifications of proteins in one or more signaling pathways, resulting in change of expression and activity of urea cycle enzymes. This hypothesis will be tested by measuring changes in protein levels and their modifications in response to varying nitrogen loads using two- dimensional gel electrophoresis, densitometry and mass spectrometry.